Complex microbial inoculant and application thereof in prevention and treatment of tobacco root rot

Abstract

The invention provides a complex microbial inoculant and application of the complex microbial inoculant in prevention and treatment of tobacco root rot, and belongs to the technical field of biocontrol inoculants. The biocontrol microbial inoculum disclosed by the invention is prepared by mixing and fermenting bacillus megatherium T482 and bacillus safensis YN-LJG-4. According to the invention, the two bacteria are subjected to co-fermentation, such that the composite bacterial agent is prepared. The bacillus megaterium T482 and the bacillus safensis YN-LJG-4 have a synergistic effect, so that the tobacco root rot can be effectively prevented and treated, and the tobacco quality can be improved. The complex microbial inoculant provided by the invention is simple in preparation method, has multiple effects, and shows a good application prospect in prevention and treatment of tobacco rhizome diseases as a biocontrol inoculant.

Description

Technical Field

[0001] The present invention relates to the technical field of biocontrol agents, and particularly to a composite bacterium agent and its application in controlling tobacco root rot disease. Background Art

[0003] Tobacco Fusarium root rot is a soil-borne disease of tobacco caused by fungi of the genus Fusarium. This disease can cause lesions in the roots of plants. Sometimes only one side is affected, causing the top to bend to one side, with small leaves. The roots of tobacco rot. When the diseased stems and roots are dissected, it can be observed that the xylem turns brown, damaging the vascular system of tobacco, resulting in difficulties in water absorption in the above-ground part and nutrient absorption in the underground part. Finally, the tobacco plants die, causing losses to tobacco.

[0004] Microbial agents have unique advantages in controlling tobacco diseases. Compared with chemical control, microbial control causes less pollution to the environment, does not produce harmful residues, can play a continuous control role, and has a relatively long-lasting control effect on pests and diseases. At present, there are many types of microbial agents. For example, the patent with the application number "202210358097.8" discloses a Bacillus velezensis RJW-5-5, which can be used for the biological control of tobacco double black diseases (black shank, root black rot); the patent with the application number "201210440492.7" discloses a Bacillus sp. strain ZY-9-13, which has a good control effect on the combined infection of tobacco root-knot nematodes and tobacco black shank pathogens. However, the microbial agents reported currently have relatively single functions and can only play a disease-resistant effect, and the growth-promoting effect on plants is not obvious. The patent with the application number "202210526304.6" discloses a microbial composite bacterium agent, which has the functions of controlling tobacco root rot, promoting the growth of tobacco, and improving the stress resistance of tobacco. However, this bacterium agent is prepared by separately fermenting three strains and then mixing the bacterial solutions, and the preparation method is relatively cumbersome. Summary of the Invention

[0005] The purpose of the present invention is to provide a composite bacterium agent and its application in controlling tobacco root rot disease, so as to solve the problems of single function of biocontrol agents and cumbersome preparation methods.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a composite bacterium agent, which is prepared by mixed fermentation of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4.

[0008] Preferably, the effective viable count in the composite bacterium agent is 1-9×10 9 cfu / mL.

[0009] The present invention also provides a method for preparing the compound microbial agent, which comprises the following steps:

[0010] S1. Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 are respectively inoculated on a seed liquid medium for independent culture, and cultured with shaking at 28-30 °C for 20-24 h;

[0011] S2. The seed liquids of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 are simultaneously inoculated into a fermentation medium, and fermented at 28-30 °C and 180-200 rpm for 30-40 h to obtain the compound microbial agent.

[0012] Preferably, the seed liquid medium is an LB medium, which comprises 9-11 g / L of peptone, 4-6 g / L of yeast extract, 9-11 g / L of NaCl and the balance of water.

[0013] Preferably, the seed liquid medium is an LB medium, which comprises 9-11 g / L of peptone, 4-6 g / L of yeast extract, 9-11 g / L of NaCl and the balance of water.

[0014] Preferably, the fermentation medium comprises the following components in the following concentrations: 3-5 g / L of sucrose, 1.5-2.5 g / L of mannitol, 8-10 g / L of yeast extract, 0.4-0.6 g / L of potassium dihydrogen phosphate, 2-3 g / L of NaCl, and 0.8-1.2 g / L of amino acid.

[0015] Preferably, the amino acid is proline.

[0016] Preferably, the inoculation amount of the seed liquid of Bacillus megaterium T482 is 1-2%, and the inoculation amount of the seed liquid of Bacillus safensis YN-LJG-4 is 2-3%.

[0017] The present invention also provides an application of the compound microbial agent in preventing and treating tobacco root rot.

[0018] The present invention also provides an application of the compound microbial agent in promoting the growth of tobacco and improving the quality of tobacco.

[0019] The present invention provides a method for preventing and treating tobacco root rot, which comprises irrigating the roots of tobacco with the compound microbial agent.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects: The biocontrol agent of the present invention is prepared by co-fermenting Bacillus megaterium T482 and Bacillus safensis YN-LJG-4. By co-fermenting the two strains, the compound microbial agent is obtained. Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 have a synergistic effect and can effectively control tobacco root rot and improve the quality of tobacco. The preparation method of the compound microbial agent of the present invention is simple and has multiple functions, showing good application prospects as a biocontrol agent in the prevention and control of tobacco root and stem diseases. Detailed implementation mode

[0021] The present invention provides a compound microbial agent, which is prepared by co-fermenting the strain Bacillus megaterium T482 and Bacillus safensis YN-LJG-4. The strain Bacillus megaterium T482 was purchased from the China Center for Type Culture Collection, and the preservation number is CCTCC M 2015754; the Bacillus safensis was purchased from the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No. 27870.

[0022] The effective viable count in the compound microbial agent of the present invention is 1-9×10 9 cfu / mL, further preferably 3-6×10 9 cfu / mL, more preferably 5×10 9 cfu / mL.

[0023] The present invention also provides a preparation method of the compound microbial agent, which includes the following steps:

[0024] S1. Respectively inoculate Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 on the seed liquid medium for independent culture, and shake culture at 28-30°C for 20-24 h;

[0025] S2. Simultaneously inoculate the seed liquids of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 into the fermentation medium, and ferment at 28-30°C and 180-200 rpm for 30-40 h to obtain the product.

[0026] The present invention first inoculates Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 in the slant medium in the strain preservation tube for culture to obtain slant cultures; the slant medium is preferably LB medium, the culture time is 20-24 h, further preferably 21-23 h, more preferably 22 h; the culture temperature is 28-30°C, further preferably 28.5-29.5°C, more preferably 29°C.

[0027] In the present invention, slant cultures of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 are separately picked and inoculated onto a seed medium for independent culture. The culture time is 20 - 24 h, further preferably 21 - 23 h, and more preferably 22 h; the culture temperature is 28 - 30 °C, further preferably 28.5 - 29.5 °C, and more preferably 29 °C.

[0028] The seed medium is LB medium, which includes 9 - 11 g / L of peptone, 4 - 6 g / L of yeast extract, 9 - 11 g / L of NaCl and the balance of water; the concentration of peptone in the seed medium is further preferably 9.5 - 10.5 g / L, and more preferably 10 g / L; the concentration of yeast extract is further preferably 4.5 - 5.5 g / L, and more preferably 5 g / L; the concentration of NaCl is further preferably 9.5 - 10.5 g / L, and more preferably 10 g / L. The seed culture is a shaking culture, and the shaking frequency is 180 - 220 rpm, further preferably 190 - 210 rpm, and more preferably 200 rpm.

[0029] In the present invention, the seed liquors of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 are simultaneously inoculated into a fermentation medium for fermentation. The inoculation amount of the seed liquor of Bacillus megaterium T482 is 1 - 2%, further preferably 1.3 - 1.7%, and more preferably 1.5%; the inoculation amount of the seed liquor of Bacillus safensis YN-LJG-4 is 2 - 3%, further preferably 2.2 - 2.8%, and more preferably 2.5%; the fermentation temperature is 28 - 30 °C, further preferably 28.5 - 29.5 °C, and more preferably 29 °C; the fermentation time is 30 - 40 h, further preferably 32 - 38 h, and more preferably 35 h; the fermentation is accompanied by stirring, and the stirring speed is 180 - 200 rpm, further preferably 185 - 195 rpm, and more preferably 190 rpm. The fermentation medium includes the following components at the following concentrations: 3 - 5 g / L of sucrose, 1.5 - 2.5 g / L of mannitol, 8 - 10 g / L of yeast extract, 0.4 - 0.6 g / L of potassium dihydrogen phosphate, 2 - 3 g / L of NaCl, 0.8 - 1.2 g / L of amino acids; the concentration of sucrose in the fermentation medium is further preferably 3.5 - 4.5 g / L, and more preferably 4 g / L; the concentration of mannitol is further preferably 1.7 - 2.2 g / L, and more preferably 2 g / L; the concentration of yeast extract is further preferably 8.5 - 9.5 g / L, and more preferably 9 g / L; the concentration of potassium dihydrogen phosphate is further preferably 0.45 - 0.55 g / L, and more preferably 5 g / L; the concentration of NaCl is further preferably 2.2 - 2.8 g / L, and more preferably 2.5 g / L; the concentration of amino acids is further preferably 0.9 - 1.1 g / L, and more preferably 1 g / L.

[0030] The present invention also provides the application of the compound microbial agent in preventing and treating tobacco root rot.

[0031] The present invention also provides the application of the compound microbial agent in promoting the growth of tobacco and improving the quality of tobacco.

[0032] The present invention also provides a method for preventing and treating tobacco root rot, which is characterized in that after diluting the compound microbial agent, root irrigation treatment is carried out on tobacco. In the diluted bacterial liquid, the effective viable count is 1-9×10 7 cfu / mL, further preferably 3-6×10 7 cfu / mL, and more preferably 5×10 7 cfu / mL.

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] The preparation method of the culture medium in the embodiments is as follows:

[0035] Slant medium: Add 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and 15 g / L agar to distilled water. After stirring until completely dissolved, adjust the pH to 6.8, make up the volume, and sterilize at 120°C for 20 min to obtain.

[0036] Seed liquid medium: Add 10 g / L peptone, 5 g / L yeast powder, and 10 g / L NaCl to distilled water. After stirring until completely dissolved, adjust the pH to 7.2, make up the volume, and sterilize at 120°C for 20 min to obtain.

[0037] Fermentation medium: Add 4 g / L sucrose, 2 g / L mannitol, 9 g / L yeast powder, 0.5 g / L, 2.5 g / L, and 1 g / L proline to distilled water. After stirring until completely dissolved, adjust the pH to 7.1, make up the volume, and sterilize at 120°C for 20 min to obtain.

[0038] Example 1

[0039] (1) Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 in the strain preservation tubes were respectively inoculated into the slant medium and cultured at 28°C for 24 h to obtain slant cultures;

[0040] (2) The slant cultures of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 were respectively picked and inoculated onto the seed liquid medium, and cultured at 28°C and 180 rpm for 24 h to obtain the seed liquids of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4;

[0041] (3) Inoculate the seed solutions of *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4 into the fermentation medium simultaneously. The inoculation amount of *Bacillus megaterium* T482 is 1%, and the inoculation amount of *Bacillus safensis* YN-LJG-4 is 3%. Ferment at 28 °C and 180 rpm for 40 h to obtain the composite microbial agent. By plate counting, the viable cell count in the composite microbial agent is 5.8×10 9 cfu / mL.

[0042] Example 2

[0043] (1) Inoculate *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4 in the slant medium from the strain preservation tubes respectively, and culture at 29 °C for 22 h to obtain slant cultures;

[0044] (2) Pick the slant cultures of *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4 respectively, inoculate them on the seed solution medium, and culture by shaking at 29 °C and 200 rpm for 22 h to obtain the seed solutions of *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4;

[0045] (3) Inoculate the seed solutions of *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4 into the fermentation medium simultaneously. The inoculation amount of *Bacillus megaterium* T482 is 1.5%, and the inoculation amount of *Bacillus safensis* YN-LJG-4 is 2.5%. Ferment at 29 °C and 190 rpm for 35 h to obtain the composite microbial agent. By plate counting, the viable cell count in the composite microbial agent is 7.2×10 9 cfu / mL.

[0046] Example 3

[0047] (1) Inoculate *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4 in the slant medium from the strain preservation tubes respectively, and culture at 30 °C for 20 h to obtain slant cultures;

[0048] (2) Pick the slant cultures of *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4 respectively, inoculate them on the seed solution medium, and culture by shaking at 30 °C and 220 rpm for 20 h to obtain the seed solutions of *Bacillus megaterium* T482 and *Bacillus safensis* YN-LJG-4;

[0049] (3) Inoculate the seed solutions of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 into the fermentation medium simultaneously. The inoculation amount of Bacillus megaterium T482 is 2%, and the inoculation amount of Bacillus safensis YN-LJG-4 is 2%. Ferment at 30 °C and 200 rpm for 30 h to obtain the composite microbial agent. By plate counting method, the viable cell count in the composite microbial agent is 6.4×10 9 cfu / mL.

[0050] Comparative Example 1

[0051] Differing from Example 1, only Bacillus megaterium T482 was inoculated for fermentation, and the inoculation amount was 4%.

[0052] Comparative Example 2

[0053] Differing from Example 1, only Bacillus safensis YN-LJG-4 was inoculated for fermentation, and the inoculation amount was 4%.

[0054] Comparative Example 3

[0055] Differing from Example 1, mannitol was not added to the fermentation medium, and an equal amount of sucrose was used for substitution.

[0056] Experimental Example 1. Quantitative determination of IAA

[0057] Salkowski's reagent: 1 mL of 0.5 mol / L FeCl 3 and 49 mL of 35% perchloric acid.

[0058] Centrifuge the fermentation broths of Examples 1 to 3 and Comparative Examples 1 to 3 at 10000 r / min for 5 min. Take 100 μL of the supernatant and add it to a 96-well plate. Mix it evenly with an equal amount of Salkowski's reagent, and let it stand at room temperature for 30 min. Prepare a standard curve using IAA standard products with different concentrations, and measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 530 nm. The measurement results are shown in Table 1.

[0059] Table 1 IAA concentrations in the fermentation broths of each group

[0060] Treatment group IAA concentration (mg / L) Example 1 315.61 Example 2 327.38 Example 3 318.26 Comparative Example 1 245.21 Comparative Example 2 0 Comparative Example 3 283.15

[0061] As can be seen from Table 1, the co-fermentation of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 can improve the ability of Bacillus megaterium T482 to produce IAA, indicating that the two have a synergistic effect. At the same time, from Comparative Example 3, it can be seen that adding sucrose and mannitol as carbon sources to the fermentation medium simultaneously can promote the secretion of IAA by Bacillus megaterium T482.

[0062] Experimental Example 2. Determination of antibacterial rate

[0063] In a sterile operating table, inoculate a pathogen cake of tobacco root rot pathogen with a diameter of 0.5 cm on a PDA plate medium. Drop 10 μL of the fermentation broths of Examples 1 to 3 and Comparative Examples 1 to 3 at a distance of 2.5 cm from the pathogen cake. The control group drops an equal amount of deionized water. After culturing in an inverted position in an incubator at 28 °C for 15 days, count the antibacterial rate. The results are shown in Table 2.

[0064] Antibacterial rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%

[0065] Table 2 Antibacterial rates of fermentation broths of each group

[0066] Treatment group Bacteriostatic rate (%) Example 1 91.83 Example 2 93.26 Example 3 91.15 Comparative Example 1 0 Comparative Example 2 56.69 Comparative Example 3 78.12

[0067] As can be seen from Table 1, the co-fermentation of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 can improve the antibacterial ability of Bacillus safensis YN-LJG-4, indicating that they have a synergistic effect. At the same time, as can be seen from Comparative Example 3, adding sucrose and mannitol as carbon sources in the fermentation medium simultaneously can promote the generation of antibacterial metabolites in Bacillus safensis YN-LJG-4.

[0068] Experimental Example 3. Effects on the control effect and yield of tobacco root rot in the field

[0069] An experiment was carried out using naturally occurring disease soil in the field. The experiment was carried out in a tobacco planting base in Kuandian County, Dandong. The tobacco test variety was Yunyan 87. The field was divided into 21 plots, each plot being 100 m 2 . A randomized block design was used, with protective rows set around. The tobacco was transplanted on February 8, 2023. The fermentation broths in Examples 1 to 3 and Comparative Examples 1 to 3 were diluted 100 times and each was irrigated into the roots once during the tobacco transplanting period and the rosette stage, 600 mL per plant each time. The control group was irrigated with an equal amount of water. Each group had three replicates. On the 15th day after the last root irrigation, the disease index and control effect were counted. Ten points were selected in each plot, and 10 tobacco plants were selected at each point for statistics. The results are shown in Table 3.

[0070] The disease grading of tobacco root rot was carried out according to the tobacco industry standard GB / T 23222—2008.

[0071] Grade 0: Disease-free, normal plant growth;

[0072] Grade 1: The plant grows basically normally or is slightly dwarfed, a few roots are necrotic and black, and the middle and lower leaves are chlorotic (or discolored);

[0073] Grade 3: The height of the diseased plant is one-fourth to one-third shorter than that of the healthy plant, or half of the roots are necrotic and black, one-half to two-thirds of the leaves are wilted, and the middle and lower leaves have slightly dry tips and edges;

[0074] Level 5: Diseased plants are two-thirds to one-half shorter than healthy plants. Most of the root rings are necrotic and black. More than two-thirds of the leaves are wilted, with obvious dry tips and dry edges.

[0075] Level 7: Diseased plants are more than one-half shorter than healthy plants. All the leaves of the whole plant are withered. All the roots are necrotic and black, and the secondary roots near the ground surface are significantly damaged.

[0076] Level 9: All the leaves of the whole plant are dead.

[0077]

[0078] Control efficacy = (disease index in treatment area - disease index in control area) / disease index in control area × 100%

[0079] Table 3 Disease indices and control efficacies of tobacco in each treatment group

[0080] Treatment group Disease index Control effect (%) Example 1 5.13 85.00 Example 2 4.91 87.74 Example 3 5.02 85.32 Comparative Example 1 34.15 0.001 Comparative Example 2 15.37 55.05 Comparative Example 3 10.14 70.34 Control 34.19 --

[0081] The control efficacy of tobacco is the same as the trend of the bacteriostatic rate in Experiment 2. The co-fermentation of Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 can improve the control efficacy of Bacillus safensis YN-LJG-4 against tobacco root rot.

[0082] After the tobacco is harvested, the yields of tobacco in each plot are counted, and the average value is calculated. Then, the average yield of each group is converted into the yield per mu. The results are shown in Table 4.

[0083] Table 4 Yields per mu (dry weight) of tobacco in each treatment group

[0084]

[0085]

[0086] As can be seen from Table 4, the compound bactericide of the embodiment of the present invention can significantly improve the yield per mu of tobacco, and the effect is better than that of the control group and the comparative example.

[0087] As can be seen from the above embodiments, the present invention provides a compound bactericide and its application in preventing and treating tobacco root rot. Bacillus megaterium T482 and Bacillus safensis YN-LJG-4 in the compound bactericide have a synergistic effect, can effectively prevent tobacco root rot, and improve the tobacco yield.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composite bacterial agent, characterized in that: It is prepared by mixed fermentation of Bacillus megaterium T482 and Bacillus saffron YN-LJG-4.

2. The composite bacterial agent according to claim 1, characterized in that The effective number of live bacteria in the composite bacterial agent is 1 to 9×10 9 cfu / mL.

3. The method for preparing the composite bacterial agent according to claim 1 or 2, characterized in that: The steps include: S1. Bacillus megaterium T482 and Bacillus shafusus YN-LJG-4 were inoculated on seed liquid medium and cultured independently at 28-30°C with shaking for 20-24h; S2. The seed liquid of Bacillus megaterium T482 and Bacillus shafusus YN-LJG-4 are inoculated into the fermentation medium at the same time, and fermented at 28-30° C. and 180-200 rpm for 30-40 hours to obtain the product.

4. The preparation method according to claim 3, characterized in that: The seed liquid culture medium is LB culture medium, which includes 9-11 g / L of peptone, 4-6 g / L of yeast powder, 9-11 g / L of NaCl and the balance of water.

5. The preparation method according to claim 3, characterized in that: The fermentation medium comprises the following components in concentrations: 3-5 g / L sucrose, 1.5-2.5 g / L mannitol, 8-10 g / L yeast powder, 0.4-0.6 g / L potassium dihydrogen phosphate, 2-3 g / L NaCl, and 0.8-1.2 g / L amino acids.

6. The preparation method according to claim 5, characterized in that: The amino acid is proline.

7. The preparation method according to claim 3, characterized in that: The inoculation amount of the seed solution of Bacillus megaterium T482 is 1-2%, and the inoculation amount of the seed solution of Bacillus saffron YN-LJG-4 is 2-3%.

8. Use of the composite bacterial agent according to claim 1 or 2 in preventing and treating tobacco root rot.

9. Use of the composite bacterial agent according to claim 1 or 2 in promoting tobacco growth and improving tobacco quality.

10. A method for preventing and treating tobacco root rot, characterized in that: The composite bacterial agent according to claim 1 is used to perform root irrigation treatment on tobacco.

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